European Journal of Human Genetics (2003) 11, 23 – 29 ª 2003 Nature Publishing Group All rights reserved 1018 – 4813/03 $25.00 www.nature.com/ejhg ARTICLE Cystathionine b-synthase polymorphisms and hyperhomocysteinaemia: an association study Karin JA Lievers1, Leo AJ Kluijtmans1, Sandra G Heil1, Godfried HJ Boers2, Petra Verhoef4, Martin den Heijer3, Frans JM Trijbels1 and Henk J Blom*,1 1Laboratory of Pediatrics and Neurology, University Medical Center Nijmegen, The Netherlands; 2Department of Internal Medicine, University Medical Center Nijmegen, The Netherlands; 3Department of Internal Medicine, Division of Endocrinology, University Medical Center Nijmegen, The Netherlands; 4Wageningen Center for Food Sciences and Division of Human Nutrition and Epidemiology, Wageningen University, Wageningen, The Netherlands Hyperhomocysteinaemia is generally accepted as an independent and graded risk factor for both arterial occlusive disease and venous thrombosis. The only way of homocysteine degradation is conversion to cysteine in the transsulfuration pathway in which the regulating step is catalysed by cystathionine b- synthase (CBS). Mild impairment of CBS function could therefore affect homocysteine concentration, in particular after methionine loading, and consequently cardiovascular disease (CVD) risk. We analysed two silent polymorphisms and one short tandem repeat in the CBS gene (ie 699C?T, 1080C?T and – 5697 (GT) STR) as genetic markers potentially in linkage disequilibrium with a functional polymorphism. We assessed their association with fasting and post-methionine load homocysteine in 190 patients with arterial occlusive disease, and in 381 controls. No differences in CBS genotype frequencies between cases and controls were found, nor was a particular CBS genotype associated with an elevated risk of arterial occlusive disease. Although we did find a high rate of linkage disequilibrium between the two single nucleotide polymorphisms and the GT STR, none of the genotypes defined by the three CBS variants studied showed an association with elevated fasting, post-load or increase upon methionine loading homocysteine concentrations. In conclusion, we did not find any indication that genetic variation in the CBS gene is associated with increased homocysteine concentrations. European Journal of Human Genetics (2003) 11, 23 – 29. doi:10.1038/sj.ejhg.5200899 Keywords: cystathionine b-synthase (CBS); homocysteine; polymorphism; cardiovascular disease; association study; linkage disequilibrium Introduction methionine metabolism, may either be irreversibly Established risk factors for cardiovascular disease (CVD) degraded to cysteine via cystathionine in the transsulfura- include elevated blood cholesterol, hypertension, diabetes tion pathway or remethylated to methionine. The mellitus and tobacco smoking. A modest elevation of plas- remethylation by methionine synthase requires 5-methyl- ma homocysteine concentration, commonly referred to as tetrahydrofolate for methyl donation and vitamin B12 for hyperhomocysteinemia, is generally,1,2 though not univer- methyl transport, while the remethylation by betaine- sally3,4 accepted as an independent and graded risk factor homocysteine-methyltransferase requires betaine as methyl for both arterial occlusive diseases and venous thrombo- donor. The regulating step in the transsulfuration pathway sis.5,6 Homocysteine, an intermediate sulfur amino acid in is catalysed by cystathionine b-synthase (CBS), a pyridoxal 5’-phosphate-dependent enzyme. The inherited metabolic *Correspondence: Dr HJ Blom, Laboratory of Pediatrics and Neurology, disorder CBS deficiency is acknowledged to be the most University Medical Center Nijmegen, PO Box 9101, 6500 HB, Nijmegen, frequent cause of homocystinuria in humans.7 The Netherlands. Tel: +31 24 3613469; Fax: +31 24 3618900; 8 E-mail: [email protected] In 1985, Boers et al postulated heterozygosity for CBS Received 4 June 2002; revised 7 August 2002; accepted 26 August 2002 deficiency as a major cause of hyperhomocysteinaemia in CBS association study KJA Lievers et al 24 CVD patients. This was based on the decreased CBS activ- be in linkage disequilibrium with a functional polymorph- ities they found in extracts of cultured fibroblasts of ism. We analysed two silent single nucleotide hyperhomocysteinaemic vascular disease patients and their polymorphisms (699C?T, 1080C?T) and a short tandem increased post-methionine loading homocysteine concen- repeat (STR) – 5697(GT),22 in the CBS gene in 190 vascular trations, both resembling those of obligate heterozygotes disease patients and in 381 population-based controls. We for CBS deficiency. Similar results were described by Clarke report the allele frequencies and genotype distributions of et al.9 Both studies however, do not reconcile with an these polymorphic variants in both study groups, and earlier observation by Mudd et al,10 who described a similar describe their association with fasting, post-methionine incidence of heart attacks or strokes in parents and grand- load and delta (ie increase upon methionine loading) plas- parents of homocystinuric children compared with ma homocysteine concentrations. Also, the extent of relatives of children with either new-mutation achondro- linkage disequilibrium between the several CBS variants plastic dwarfism or with impaired phenylalanine are calculated. metabolism. Furthermore, the finding of decreased CBS activities in cultured fibroblasts from hyperhomocysteinae- Materials and methods mic vascular disease patients in the range of obligate Study population heterozygotes could not be reproduced by us,11,12 and still We studied 190 patients with coronary, peripheral or cere- needs clarification. In addition to fasting homocysteine bral vascular disease; a total of 130 cases were recruited concentrations, also post-methionine load homocysteine from a cohort of patients who underwent coronary angio- concentrations were found to be correlated among family graphy in the Zuiderziekenhuis Hospital in Rotterdam, members of patients with hyperhomocysteinaemia and The Netherlands.18 Reasons for angiography were myocar- vascular disease.13 – 15 So, it can be concluded that hyper- dial infarction or angina pectoris and cases were defined homocysteinaemia is, at least partially, genetically based. as those having 590% occlusion in one and 540% occlu- Still, elevated post-methionine load homocysteine concen- sion in one additional coronary artery. The other 60 trations are associated with increased CVD risk,16 – 18 but patients had documented premature cardiovascular disease its genetic determinants remain obscure. of which 10 patients had suffered from myocardial infarc- Over 100 mutations have been described in the CBS gene tion, 32 from cerebral arterial occlusive disease and 18 in classical homocystinuria patients.19 A 68 bp insertion in from peripheral arterial occlusive disease.12 exon 8, first reported as a causal mutation in an Italian The control group consisted of 381 controls, of whom patient, was found to be polymorphic and not to be asso- 101 were included from the general population in Rotter- ciated with plasma homocysteine concentrations.20,21 No dam and comprised subjects with no history of other potentially functional polymorphisms associated with cardiovascular disease18 and 280 were recruited from a elevated homocysteine levels were detected in the CBS gene general practice in The Hague, The Netherlands to take part in the analyses of the genetic basis of homocystinuria. in a health survey.25 However, most studies performed thus far only examined the coding region of the CBS gene, leaving the non-coding Biochemical parameters regions unexplored. After an overnight fast, blood was collected in EDTA tubes Very recently, we described an inverse correlation for measurement of fasting total homocysteine. The between the number of tandem repeats defined by a EDTA-blood samples were placed on ice immediately, and 31 bp VNTR (variable number of tandem repeats),22 and centrifuged for 10 min at 3000 g with minimal delay. All CBS enzyme activities in fibroblasts.23 Furthermore, a posi- individuals were subjected to a standardised methionine tive association was observed between the number of repeat loading test.26 Briefly, L-methionine (0.1 g/kg body weight) units and post-methionine load plasma homocysteine was dissolved in 200 mL orange juice and administered concentrations.23 orally. All study subjects received a standardised low protein Genetic association studies are widely used for the iden- breakfast and luncheon, and were asked not to consume tification of candidate genes of complex, ie non-mendelian any milk-containing beverages. After 6 h, another blood traits.24 In such studies, the association is investigated sample was drawn for assessment of the post-methionine between a phenotype and genomic markers close to or, load homocysteine concentration. Total homocysteine was preferably, within the gene of interest. Such a marker in measured in plasma using a high-performance liquid chro- itself is not necessarily functionally related to the pheno- matography (HPLC) procedure, with reverse phase type but may be in linkage disequilibrium with a separation and fluorescence detection, as described by Te functional variant. Poele-Pothoff et al.27 All homocysteine measurements were In the present study, we investigated the potential invol- conducted at the University Medical Center Nijmegen, The vement of genetic variants in non-coding regions or of Netherlands, and fasting and post-load